COMMUNICATIONS TO THE EDITOR
April 5 , 1958
been highly purified by H i l m ~ e . The ~ degradation of compounds of the type T,P,-, by this preparation was studied. It was found that, again, all the lower homologs were formed successively and that thymidine appeared only toward the end. However, the mononucleotide which accumulated was thymidine 3’-phosphate, as in the ribonucleotide series.* These results show that the action of this enzyme as well is stepzise but begins from the end bearing the 5‘-hydroxyl group. TABLE I The reaction mixture consisted of X trihydroxymethylaminomethane, p H 8.9 (10 pl.), TjPl (14 qptical density units measured a t 267 mp), venom diesterase (31 pg. in 7 pl.) and water (20 pl,). .Iliquots (6 pl,) were mixed with glacial acetic acid (1 pl.) and chromatographed in isopropyl alcoholammonia-water (7-1-2). T h e figures are t h e yoof the total optical density in each aliquot. Compound b>i
2 5 15 30 60
TsP; 0.05
36 10
9
i
4
T ~ P J TAP? 0.065 0.24 Time, min.
34 21 0 4 3
5 19 11 3 1
T>P 0.48
0 8 14 10 3
T 0.70
0 0
2 6 13
TP 0.18
25 42 58 70 76
It should be noted that both the snake venom and spleen phosphodiesterase preparations hydrolyzed, even if slowly, cyclic oligonucleotides1 in which the 3‘-hydroxyl group is involved in an ester linkage with the 5’-phosphate group a t the other end of the chain. It is therefore possible for these preparations to attack a t a point within a polynucleotide chain (cf. ref. 3). The stepwise and complementary action of the two phosphodiesterases is strikingly reminiscent of the two proteolytic enzymes, carboxypeptidase and (leucine) aminopeptidase, which degrade polypeptide chains from the opposite ends, and is promising for the structural and sequential analysis of polynucleotides. Acknowledgment.-This work has been supported by grants from the National Cancer Institute of the National Institutes of Health, U. S. Public Health Service, and the National Research Council of Canada, Ottawa. (7) R. J . Hilmoe, unpublished work.
We are grateful for a gift of
1771
received support from Umbarger, et aZ.4 I n the present communication we wish t o describe the properties of a cell-free preparation from Saccharomyces cerevisiae which converts acetolactic acid t o a-ketoisovaleric acid, the keto analog of valine. Fresh baker’s yeast (Fleischmann) was disrupted in 0.02 Ad phosphate buffer, pH 7.2, by means of a high-speed refrigerated centrifuge shaker described by Shockman, et a1.6 The crude, cell-free supernatant obtained after centrifugation a t 80,000 g was either used as such or was dialyzed against cold distilled water for 18-20 hours. The enzyme preparations were incubated a t 37” with acetolactic acid, synthesized as described by Krampitz.6 After 4 hours, the solution was deproteinized with tungstic acid, and the 2,4-dinitrophenylhydrazones of the keto acids were prepared, extracted and chromatographed on paper, essentially according to the procedure of Cavalini, et al? Results of a single, typical experiment, illustrative of the properties of this system, are shown in Table I. Mixtures of undialyzed extract and acetolactic acid produced a strong hydrazone spot, not observed in the absence of either enzyme or substrate. The identity of this material with the hydrazone of a-ketoisovaleric acid was established by identical Rf values in four different solvents, by identical absorption spectra over the wave length range of 350 to 700 my, and by reduction of material isolated from a strip chromatogram, according to Towers, et a1.,8 to a substance identical chromatographically with valine in three different solvent systems. TABLE I ESZYMATIC CONVERSION OF CY-ACETOLACTATE ‘ro CY-KETOISOVALERATE Each tube contained 80 pmoles potassium phosphate buffer, pH 8.0, and where indicated in the table, 0.05 mg. T P S , 0.5 mg. DPX, 1.0 mg. ATP, 27 pmoles neutralized acetolactate, pH 8.0, 27 pmoles K acetate, 27 pmoles ethanol, 15 pmoles magnesium sulfate, and 0.3 ml. of dialyzed or undialyzed enzyme, containing approximately 4 mg. protein (by ultraviolet absorption) and representing 0.15 g. of fresh yeast. Total volume was 1.6 ml. and incubation was conducted 4 hours in air a t 37”. Enzyme preparation
Acetolactate
+ + + + + + +
Cofactor(s)
a1Cetoisova1:rate micromoles
1.28 Crude this preparation. TPAT 2.10 Crude (8) L . A. Heppel and R. J. Hilmoe in “Methods in Enzymology,” Vol. 11, Academic Press Inc., New York, N. Y., 1955, p . 565. 0.06 Dialyzed Dialyzed Crude heated 1.83 BRITISHCOLUMBIA RESEARCH COUNCIL USIVERSITYOF BRITISHCOLUMBIA W.E. RAZZELL Dialyzed DPS 0.14 VANCOUVER 8, B. C., CASADA H. G. KAORASA Dialyzed D P S , -ITP, M g + 2.19 RECEIVED JANUARY 24, 1958 TPN 2.97 Dialyzed Dialyzed’ acetate +ethanol TPS 0 02 This was run t o check the effects of acetate and ethano ENZYME STUDIES ON THE BIOSYNTHESIS OF present as a result of hydrolysis of the ethyl acetoxyncetoVALINE IN YEAST lactate.
Sir: I n previous r e p ~ r t s l - based ~ on isotope tracer data, a mechanism for the biosynthesis of valine was proposed, which involved a-acetolactic acid as an intermediate. This hypothesis has recently (1) M. Strassman, A J. Thomas and S. Weinhouse, TEIISJ O U R N A L 75, 5135 (1953); 7 7 , 1261 (1955). (2) E . A . Bdelberg, ibid.,76, 4241 (1954). (3) M. Strassman, A. J. Thomas, L . A. Locke a n d S. Weinhouse, i b i d . , 76, 4241 (1954).
The values given in Table I were obtained b y elution of the a-ketoisovaleric acid hydrazone spots (4) H. E. Umbarger, B. Brown a n d E. J . Eyring, i b i d . , 79, 2980 (19.57). ( 5 ) G. D. Shockman, J. J. Kolb and G. Toennies, B i o c h i m . B i o g h y s . A c t a , 2 4 , 203,(1957). (6) L . 0. Krampitz, A v c h . B i o c h e m . , 17, 8 1 (1948). (7) D. Cavalini a n d N. Frontali, B i o c h i m . B i o p h y s . A r i a , 13, 439 (1954). (8) G. H. N. Towers, J . F. Thompson a n d F. C. Steward, THIS JOURNAL, 76, 2392 (1956).
1x3
COMMITXICATIONS TO THE EDITOR
Vol. 80
and measurement of their absorption at 510 mp.7 h mixture of undialyzed enzyme and acetolactic acid produced 1.3 pmoles of a-ketoisovaleric acid, and the yield was almost doubled by T P N adclition. Crude extract or substrate alone, or dialyzed extract with substrate yielded only negligible arnounts. -1ddition of boiled crude extract, T P N , or DPN arid ATP restored activity of the dialyzed extract. These data demonstrate that acetolactate is a precursor of valine in S. cereuisiae, and suggest that T P K (probably T P N H ) is involved with this transformation. Further efforts are proceeding for purification and fractionation of this enzyme system and for identification of several other keto acids which appeared in the chromatogram^.^
ing known partial pressures of oxygen. Oxygen, a t a known pressure, was introduced into a stainless steel bomb containing cells with polonium solutions for high pressure studies. Oxygen concentrations were calculated from the data of Zoss, Suciu and Sibbitt.' Suitable corrections were made for the dark reaction and oxidation at atinospheric pressures. The change in ferric ion concentration was followed spectrophotometrically and the rate of energy absorption in the solutions was obtained from the rate of Po21odisintegration. This rate was measured by absolute counting in a calibrated pulse ionization chamber. The full energy of the 5.3 MeV. alpha particle was assumed to have been expended within the solution. G(Fe +++) increases rapidly from its air-free value of 3.7 to 3.2 for air-saturated solutions (2.2 X (9) This uork was supported by grants from t h e National Science -11 02).A4bove oxygen concentrations of Foundation. T h e American Cancer Society, and t h e Kational Institutes of Health. l o p 3 31, however, G(Fe+++) is nearly a linear function of the logarithm of the oxygen concentraTHEINSTITL-TE FOR CASCERRESEARCH AND MURRAY STRASSXAK tion. The highest experimental G(Fe++' ) value LASKES.4C HOSPITA4LRESEARCH IYSTITUTE obtained in this work was 9.1 f 0.4 at 1903 p.s.i. JENXIE B. SHATlOX PI?II.ADELPHIA, PEXSSYLVAKIA MILTOSE. CORSEY oxygen. The increase in G(Fe+++) with oxygen concenSIDNEY U'EISHOUSE tration is attributed to a competition of reaction RECEIVED JAYKART 30, 1933 (1) with reactions ( 3 ) , ( 3 ) , and (4) in the mechanism of the Fricke ferrous sulfate d ~ s i m e t e r . ~ THE EFFECT OF OXYGEN O N FERRIC ION YIELDS I N H + 0 2 = HOn (1) AQUEOUS SOLUTIONS CONTAINING POLONIUM H + H = Hz (2) Sir: 9 revised G(Fef++) yield of 5.2
* 0.15 has been
obtained for a-particles from dissolved Pozlo in the aerated Fricke ferrous sulfate dosimeter. The previous value of about 6.0 reported by various worker^',^,^ is probably too high. Other lower ~ a l u e s ~ .have j * ~ been reported recently. G(Fe +&+) has been found to vary from 3.7 f 0.2 in air-free solutions t o values approaching 10 at oxygen concentrations above 0.1 AT. Solutions studied were 3 m M in FeS04, 1 r n X in XaC1, 0.S N in HzS04and contained on the average 0.4 mc./ml. of Poz1". G(Fe+++) was independent of dose rate between 0.01 and 0.90 mc./ml. Oxygen pressures below 1 atmosphere were obtained by evacuating the samples and reintroduc(1) S . Xiller and J . Wilkinson, T r a n s . Favaday SOC.,5 0 , 690 (1954). (2) 1%'. R . AIcDonell and E. J . H a r t , TIXISJ O U R N A L , 76, 2 1 2 1 (19.54). (3) hf. Haissinsky a n d M. C. Anta, Compl. rend., 836, l l C l (1953). (4) W. R. Henke, P. K. Kruys and P. P . Lifland, Project Report for t h e School of S u c l e a r Science a n d Engineering, (Argonne h'ational Laboratory) 2nd Sess'on, June 1, 1956. (3) .4.0. Allen, I n l . C o n f . o i t Peacefid Uses of Afotiiir E n e v g y , Vol. 7 , 813, United Nations N. Y..1956. ( i ; ) M , Ixfort. Com,bl. rend., 245, 1 0 2 3 (1987).
H + OH = + H T y- F e + + = H P + F e + + + H20
(3)
(4 IXTorkwith higher than 3 mill initial ferrous ion concentration [ (Fe++)o] indicates a marked dependence of G ( F e r f + ) on (Fe+f)ofor oxygen concentrations in the regions of to lo-' ill. In contrast, at atmospheric pressures the ferric ion yield is much less sensitive to (Fe++)". Further work is being carried out on the effect of ferrous ion concentration and on the effect of pressures above 2000 p s i . The author wishes to express his gratitude to Drs. E. J. Hart, N. Miller and H. Fricke for valuable discussions and advice given during the course of this research which was carried out under the auspices of the United States Atomic Energy Cornmission a t Argonne National Laboratory. H
CHEMISTRY DEPARTMENT UVIVERSITY OF ROCHESTER CONRAD S ROCHESTER 20, S. Y . RECEIVED JAYUARY 31, 19%j&
TRUmORE
(7) A. Zoss. S. h'. Sucii:, W. L. Sihbitt, Tvnnr A m . S o r . M p c h . Eng., 7 6 , G!) (1%74),
